Disclosed herein are methods for large-scale, high-throughput identification of protein-protein interactions and the topologies thereof under physiologically relevant conditions. In one aspect, the disclosure provides methods for identifying one or a plurality of interacting peptides within a biological system comprising obtaining a population of proteins cross-linked with a cleavable protein interaction reporter (PIR) cross-linker, cleaving the PIR crosslinker to produce released peptides and cleaved reporter ions, and analyzing the population of released peptides to identify interacting peptides. Also disclosed are methods for identifying candidate drug compounds, as well as methods of data processing and visualization of protein-protein interactions.
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1. A method for identifying at least two different interacting peptide pairs within a biological system comprising: (a) obtaining a sample comprising a population of cross-linked precursor peptides produced by digestion of a population of proteins cross-linked with a cleavable protein interaction reporter (PIR) cross-linker; (b) subjecting the sample to mass spectrometry (MS) to: (i) produce a population of precursor ions, and (ii) generate a 1° mass spectrum used to determine the charge states and masses of precursor ions within the population of precursor ions; (c) selecting, from within the 1° mass spectrum, a precursor ion with a charge state equal to or greater than a cutoff charge state; subjecting the selected precursor ion-to conditions under which the cleavable PIR cross-linker, if present in the selected precursor ion, is cleaved, thereby producing a population of released peptides and, if present in the selected precursor ion, cleaved reporter ions; and determining the masses of the released peptides and, if present in the selected precursor ion, cleaved reporter ions by tandem mass spectrometry (MS 2 ); (d) analyzing the population of released peptides to identify one or more pairs of interacting peptides, where a pair of released peptides is identified as an interacting peptide pair if the combined mass of the pair of released peptides and cleaved reporter ion, as determined by MS 2 in step (c), is equal to the mass of the selected precursor ion, as determined by MS in step (b)(ii); and (e) repeating steps (b)-(d) at least once, and until at least two different interacting peptide pairs are identified within the biological system, wherein the same cutoff charge state is used for each repetition.
This method identifies interacting peptide pairs within a biological system. It involves obtaining a sample of cross-linked peptides created from proteins that have been linked together with a special "cleavable" linker. The sample is analyzed using mass spectrometry to determine the mass and charge of the peptides. Peptides with a charge above a certain threshold are selected, and conditions are applied to break the cleavable linker. This releases the original interacting peptides and small "reporter" ions. The masses of the released peptides are measured using a second mass spectrometry step. Interacting peptide pairs are identified if the combined mass of the two released peptides plus the mass of the reporter ion equals the mass of the original cross-linked peptide. This process is repeated to find at least two different interacting peptide pairs, using the same charge threshold each time.
2. The method of claim 1 , wherein the sample comprising a population of cross-linked precursor peptides is obtained by contacting the biological system with a cleavable protein interaction reporter (PIR) cross-linker to produce cross-linked proteins, and obtaining the population of cross-linked precursor peptides therefrom.
This method identifies interacting peptide pairs by first mixing the biological system of interest with a special "cleavable" linker that binds interacting proteins together. After the proteins are linked, the method uses mass spectrometry to identify interacting peptide pairs. Specifically, a sample of cross-linked peptides is obtained by contacting the biological system with a cleavable protein interaction reporter (PIR) cross-linker to produce cross-linked proteins, and obtaining the population of cross-linked precursor peptides therefrom. The sample is then analyzed by mass spectrometry as described previously to determine which peptides are interacting.
3. The method of claim 2 , further comprising purifying and digesting the cross-linked proteins to obtain the sample comprising a population of cross-linked precursor peptides.
This method identifies interacting peptide pairs and begins by first mixing the biological system of interest with a special "cleavable" linker that binds interacting proteins together. After the proteins are linked, they are purified to remove unwanted substances. Then the linked proteins are broken down (digested) into smaller cross-linked peptides. This peptide sample is then analyzed by mass spectrometry as described previously to determine which peptides are interacting. This method builds upon identifying interacting peptide pairs by contacting the biological system with a cleavable protein interaction reporter (PIR) cross-linker to produce cross-linked proteins, and obtaining the population of cross-linked precursor peptides therefrom. The method further comprises purifying and digesting the cross-linked proteins to obtain the sample comprising a population of cross-linked precursor peptides.
4. The method of claim 2 , wherein the biological system comprises a cell, tissue, cell lysate, blood, serum, sputum, or urine.
This claim refers to a method using biological samples like cells, tissues, blood, or urine, as described in a previous part of the patent. It simply specifies the types of biological material that can be used in the method.
5. The method of claim 1 , wherein the conditions under which the cleavable PIR cross-linker is cleaved comprise collision-induced dissociation (CID).
This method identifies interacting peptide pairs using a specific way to break the "cleavable" linker. The method analyzes cross-linked peptides using mass spectrometry, and when it's time to break the linker to release the interacting peptides, a process called collision-induced dissociation (CID) is used. CID involves colliding the cross-linked peptide with an inert gas, which causes the linker to break. After collision induced dissociation (CID) is used to break the crosslinker the released peptides can then be analyzed with mass spectrometry.
6. The method of claim 1 , wherein step (d) further comprises first identifying released peptides with masses lower than partial cleavage products to create a subset of complete cleavage products, and identifying one or more pairs of interacting peptides from released peptides within the subset of complete cleavage products.
This method identifies interacting peptide pairs by adding an extra filtering step during the analysis. After breaking the cleavable linker and releasing the peptides, the method first identifies the released peptides that are smaller than partially cleaved products. These smaller peptides represent the "complete" cleavage products. From this subset of fully released peptides, the method then identifies the interacting pairs based on their mass matching the original cross-linked peptide mass.
7. The method of claim 6 , wherein identifying released peptides with masses lower than partial cleavage products comprises identifying released peptides with masses that are less than the mass of the corresponding precursor ion minus the mass of the reporter ion minus the mass of lysine stumps, wherein lysine stumps are residual modifications that remain on lysine residues after cleavage.
This method identifies interacting peptide pairs and refines the identification of "complete" cleavage products by considering the residual modifications left after breaking the linker. Specifically, when identifying released peptides with masses lower than partial cleavage products, the method ensures the released peptide's mass is less than: the original precursor ion's mass minus the reporter ion's mass minus the mass of any lysine stumps (leftover modifications on lysine residues).
8. The method of claim 1 , further comprising determining the amino acid sequences of the released peptides within the one or more pairs of interacting peptides by subjecting the released peptides within the one or more pairs of interacting peptides to conditions that cause peptide fragmentation to yield spectra that can be identified from genomic, proteomic, or other large protein sequence databases.
This method not only identifies interacting peptide pairs but also determines the amino acid sequences of the identified peptides. After finding the interacting pairs, the individual peptides are subjected to further fragmentation, generating spectra that can be matched to protein databases (genomic, proteomic, etc.). This allows the determination of the exact amino acid sequence of each peptide in the interacting pair, providing more detail about the interacting proteins.
9. The method of claim 8 , wherein the amino acid sequences of the released peptides within the one or more pairs of interacting peptides are determined by triple mass spectrometry (MS 3 ).
This method determines the amino acid sequences of interacting peptides using a specific type of mass spectrometry: triple mass spectrometry (MS3). After finding the interacting peptide pairs, MS3 is used to further fragment the released peptides and generate the spectra needed to identify their amino acid sequences by matching against protein databases. This is a specific implementation of identifying interacting peptide pairs and finding their sequences.
10. The method of claim 1 , wherein the cutoff charge state is at least +3.
This method identifies interacting peptide pairs and sets a specific minimum threshold for the charge state of the precursor ions selected for fragmentation. The method requires that the selected precursor ion has a charge state of at least +3. This means the ion has lost at least three electrons. This charge threshold helps in selecting ions that are more likely to fragment effectively and provide useful information.
11. The method of claim 1 , wherein the cutoff charge state is at least +4.
This method identifies interacting peptide pairs and sets a specific minimum threshold for the charge state of the precursor ions selected for fragmentation. The method requires that the selected precursor ion has a charge state of at least +4. This means the ion has lost at least four electrons. This charge threshold helps in selecting ions that are more likely to fragment effectively and provide useful information.
12. The method of claim 1 , wherein the cleavable PIR cross-linker comprises formula (I): (SEQ ID NO: 27) wherein X is H, succinimid-N-yl, or phthalimid-N-yl; and Y is H or a capture moiety.
This invention relates to a method for synthesizing a cleavable polyisocyanate (PIR) cross-linker with a specific chemical structure, designed for use in bioconjugation or material modification applications. The cross-linker is formulated to enable controlled cleavage under specific conditions, facilitating precise modification or release of attached biomolecules or materials. The cleavable PIR cross-linker has the chemical structure defined by formula (I), where X is selected from hydrogen (H), succinimid-N-yl, or phthalimid-N-yl, and Y is either hydrogen (H) or a capture moiety. The succinimid-N-yl or phthalimid-N-yl groups at position X provide reactive sites for conjugation to target molecules, while the capture moiety at position Y allows for selective binding or detection. The cross-linker's cleavable nature ensures that the conjugated molecules can be released under defined conditions, such as pH changes or enzymatic action, making it useful in drug delivery, biosensors, or controlled-release systems. The invention addresses the need for versatile, cleavable cross-linkers that enable reversible modifications in biochemical and material science applications.
13. The method of claim 12 , wherein the capture moiety is biotin, a hemagglutinin (HA) tag, or a polyhistidine tag.
This method identifies interacting peptide pairs and specifies the capture moiety (Y in formula I of claim 12) of the cleavable linker. The capture moiety, used for purifying the cross-linked proteins, can be biotin, a hemagglutinin (HA) tag, or a polyhistidine tag. These tags allow for easy isolation of the cross-linked proteins before mass spectrometry analysis.
14. The method of claim 12 , wherein the cleavage condition is collision-induced dissociation (CID).
This method identifies interacting peptide pairs and uses collision-induced dissociation (CID) as the specific condition to break the cleavable linker described in claim 12 (the PIR cross-linker with formula I). CID involves colliding the cross-linked peptide with an inert gas, causing the linker to break and release the interacting peptides.
15. A method of identifying a candidate compound for treating cancer comprising: (a) contacting a peptide pair from the group consisting of: (i) (SEQ ID NO: 1) FYEQFSKNIK, (SEQ ID NO: 1) FYEQFSKNIK; (ii) (SEQ ID NO: 2) FYEAFSKNLK, (SEQ ID NO: 2) FYEAFSKNLK; (iii) (SEQ ID NO: 1 FYEQFSKNIK), (SEQ ID NO: 2) FYEAFSKNLK; (iv) (SEQ ID NO: 1) FYEQFSKNIK, (SEQ ID NO: 3) KHLEINPDHPIVETLR; (v) (SEQ ID NO: 4) APFDLFENKK, (SEQ ID NO: 1) FYEQFSKNIK; (vi) (SEQ ID NO: 1) FYEQFSKNIK, (SEQ ID NO: 5) KAAALEAMK; and (vii) (SEQ ID NO: 2) FYEAFSKNLK, (SEQ ID NO: 5) KAAALEAMK; with a plurality of test compounds under conditions suitable for binding of one member of the peptide pair to the other member of the peptide pair; and (b) identifying a test compound that reduces binding of one member of the peptide pair to the other member of the peptide pair relative to a control, wherein the identified test compound is a candidate compound for treating cancer.
This method identifies potential drug candidates for treating cancer. It involves taking specific pairs of peptides (listed with specific sequence IDs: FYEQFSKNIK, FYEAFSKNLK, KHLEINPDHPIVETLR, APFDLFENKK, KAAALEAMK) and mixing them with a collection of test compounds. The goal is to find compounds that disrupt the binding between the peptides in the pair. If a compound reduces the binding compared to a control (no compound), it is considered a potential drug candidate for cancer treatment.
16. A method of identifying a candidate compound for treating an antibiotic-resistant infection comprising: (a) contacting a peptide pair comprising KINLYGNALSR (SEQ ID NO: 6) and NDIAPYLGFGFAPKINK (SEQ ID NO: 7) with a plurality of test compounds under conditions suitable for binding of one member of the peptide pair to the other member of the peptide pair; and (b) identifying a test compound that reduces binding of one member of the peptide pair to the other member of the peptide pair relative to a control, wherein the identified test compound is a candidate compound for treating an antibiotic-resistant infection.
This method identifies potential drug candidates for treating antibiotic-resistant infections. It involves taking a specific pair of peptides (KINLYGNALSR and NDIAPYLGFGFAPKINK) and mixing them with a collection of test compounds. The goal is to find compounds that disrupt the binding between the peptides in the pair. If a compound reduces the binding compared to a control (no compound), it is considered a potential drug candidate for treating antibiotic-resistant infections.
17. A method of identifying a candidate compound for treating A. baumannii infection comprising: (a) contacting a peptide pair from the group consisting of: (i) (SEQ ID NO: 8) VFFDTNKSNIKDQYKPEIAK, (SEQ ID NO: 9) MSAAEAVKEK; (ii) (SEQ ID NO: 10) TKEGR, (SEQ ID NO: 9) MSAAEAVKEK; and (iii) (SEQ ID NO: 11) LSTQGFAWDQPIADNKTK, (SEQ ID NO: 9) MSAAEAVKEK; with a plurality of test compounds under conditions suitable for binding of one member of the peptide pair to the other member of the peptide pair; and (b) identifying a test compound that reduces binding of one member of the peptide pair to the other member of the peptide pair relative to a control, wherein the identified test compound is a candidate compound for treating A. baumannii infection.
This method identifies potential drug candidates for treating *A. baumannii* infections. It involves taking specific pairs of peptides (listed with sequence IDs: VFFDTNKSNIKDQYKPEIAK, MSAAEAVKEK, TKEGR, LSTQGFAWDQPIADNKTK) and mixing them with a collection of test compounds. The goal is to find compounds that disrupt the binding between the peptides in the pair. If a compound reduces the binding compared to a control (no compound), it is considered a potential drug candidate for treating *A. baumannii* infections.
18. The method of claim 1 , wherein the MS of step (b) is performed on the sample as it elutes from a liquid chromatography (LC) column.
This method identifies interacting peptide pairs by combining liquid chromatography (LC) with mass spectrometry (MS). The sample of cross-linked peptides is first separated using LC, where different peptides elute from the column at different times. The mass spectrometry analysis is then performed on the sample as it comes off the LC column. This separation step helps to reduce the complexity of the sample and improve the identification of interacting peptide pairs.
19. The method of claim 1 , wherein steps (b)-(d) are repeated at least 10 times.
This method repeats the core steps (mass spectrometry, linker cleavage, mass analysis, peptide pairing) used to identify interacting peptide pairs at least 10 times. By repeating these steps multiple times, the method increases the chances of identifying a greater number of interacting peptide pairs within the biological system. Each repetition involves selecting a different precursor ion for fragmentation.
20. The method of claim 1 , wherein steps (b)-(d) are repeated at least 50 times.
This method repeats the core steps (mass spectrometry, linker cleavage, mass analysis, peptide pairing) used to identify interacting peptide pairs at least 50 times. By repeating these steps a large number of times, the method maximizes the chances of identifying a comprehensive set of interacting peptide pairs within the biological system. Each repetition involves selecting a different precursor ion for fragmentation.
21. The method of claim 1 , wherein the same precursor ion is selected in no more than two repetitions of steps (b)-(d).
This method identifies interacting peptide pairs and avoids repeatedly analyzing the same precursor ion. The method ensures that the same precursor ion is selected in no more than two repetitions of the core analysis steps (mass spectrometry, linker cleavage, mass analysis, peptide pairing). This helps to maximize the diversity of peptides analyzed and increases the chance of finding different interacting pairs.
22. The method of claim 1 , wherein a different precursor ion is selected each time steps (b)-(d) are repeated.
This method identifies interacting peptide pairs and ensures maximum diversity in peptide selection. Each time the core analysis steps (mass spectrometry, linker cleavage, mass analysis, peptide pairing) are repeated, a different precursor ion is selected for fragmentation. This ensures that the method explores a wide range of peptides and increases the likelihood of identifying a comprehensive set of interacting peptide pairs.
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May 21, 2014
April 4, 2017
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